I prepared liposomes loaded with an lipophilic drug. How I can separate and measure the free drug from the encapsulated drug?
Centrifugation is the most simple method for the determination of the entrapment efficiency of the liposomes.
Procedure:
Total amount of drug present :
First you take1 ml of your liposomal suspension and add methanol until liposomes are completely digested and you get clear solution. ( I normally add 1 ml of liposomes and 9 ml of methanol). Filter the solution and dilute if required and measure the total amount of drug present in the liposome suspension.
Drug present in supernatant:
Take two ml of your liposomal suspension in eppendroff tubes and centrifuge at approximately 15,000- 20,000 rpm for 1 hour. (you need to optimize the conditions) Collect the supernatant and estimate the drug present
% EE = Total amount of drug - drug present in supernatant * 100/Total amount of drug
Centrifuge the liposomal suspension. Centrifugation will cause the hydrophobic drug to settle down because of their density and size.
Dear Najmi and all,
You can use a number of different techniques for lipophilic drug, for example:
Ultrafilteration or milipore filters, of a specific pore size. where liposomes stays in the filter and the free drug pass through these small pores.
You can use deuterium oxide (Heavy water) as dispersion medium and then via bench centrifugation you can separate free drug from entrapped drug. I have recently published a paper on this method. which is;
"Proliposome powders prepared using a slurry method for the generation of beclometasone dipropionate liposomes"
https://www.researchgate.net/publication/282812596_Proliposome_Powders_Prepared_Using_A_Slurry_Method_For_The_Generation_of_Beclometasone_Dipropionate_Liposomes
Article Proliposome Powders Prepared Using A Slurry Method For The G...
I think centrifugation is your best chance. Since density and size matters, I'm pretty sure that your liposomes will be at the bottom and just analyze the supernatant. If you I can use just this small part of sedimenting equation to check which of the entities will be sedimenting faster?:
(Density(i)-Density(liquid))*D^2
Where D is the diameter of the entity i.
Good luck!
There are various techniques to measure encapsulation efficiency. You can either measure amount of drug unencapsulated and then co relate with amount of drug encapsulated or you can directly measure amount of drug encapsulated.
I use a method.
ex. Suppose you added 100 mg of drug in formulation and total content of your formulation is 1000 mg including drug.
it means 1000 mg powder contain 100 mg drug.
If your yield is 500 mg,
it means theoretically
500 mg powder contain 50 mg drug.
or
50 mg powder contain 5 mg drug which corresponds to 100 % encapsulation
In order to check it practically,
Take 50 mg of formulation, dissolve it in solvent in which your drug and polymer is soluble and measure drug content
if you get 4 mg then
use formula: Practical Drug Content × 100/ Theoretical Drug Content
Encapsulation efficiency= 4 *100/5 = 80 %
Please suggest if it is wrong or right
There are various techniques to measure encapsulation efficiency. You can either measure amount of drug unencapsulated and then co relate with amount of drug encapsulated or you can directly measure amount of drug encapsulated.
I use a method.
ex. Suppose you added 100 mg of drug in formulation and total content of your formulation is 1000 mg including drug.
it means 1000 mg powder contain 100 mg drug.
If your yield is 500 mg,
it means theoretically
500 mg powder contain 50 mg drug.
or
50 mg powder contain 5 mg drug which corresponds to 100 % encapsulation
In order to check it practically,
Take 50 mg of formulation, dissolve it in solvent in which your drug and polymer is soluble and measure drug content
if you get 4 mg then
use formula: Practical Drug Content × 100/ Theoretical Drug Content
Encapsulation efficiency= 4 *100/5 = 80 %
Please suggest if it is wrong or right
Centrifugation is the most simple method for the determination of the entrapment efficiency of the liposomes.
Procedure:
Total amount of drug present :
First you take1 ml of your liposomal suspension and add methanol until liposomes are completely digested and you get clear solution. ( I normally add 1 ml of liposomes and 9 ml of methanol). Filter the solution and dilute if required and measure the total amount of drug present in the liposome suspension.
Drug present in supernatant:
Take two ml of your liposomal suspension in eppendroff tubes and centrifuge at approximately 15,000- 20,000 rpm for 1 hour. (you need to optimize the conditions) Collect the supernatant and estimate the drug present
% EE = Total amount of drug - drug present in supernatant * 100/Total amount of drug
Normally for hydrophilic (or lipophilic) drug encapsulation in liposomes, it is useful to make a centrifugation step of the sample and separate the lipids from the supernatant. Then a UV-Vis absorbance analisys can be done, calculating the percentage of not encapsulated drug as the complement to 100. It should be the same also for lipohilic drugs, even if the compound is encapsulated inside the lipidic double layer. Generally if the absorbance of the lipidic drug is too similar to the lipid one, it is bettere to use another instrument like GC chromatography.
thank you all of for your help, but all of the suggestion is about indirect method to calculate the drug. but what about the direct method, is there any way to look for it?
A "direct" method would require you developing a specific method for yourself...in my opinion a total waste of our time. All the "indirect" methods mentioned here are extremely acceptable in the drug delivery field.
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